Structures of Five Antibiotics Bound at the Peptidyl Transferase Center of the Large Ribosomal Subunit

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Abstract

Structures of anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M bound to the large ribosomal subunit of Haloarcula marismortui have been determined at 3.0 Å resolution. Most of these antibiotics bind to sites that overlap those of either peptidyl-tRNA or aminoacyl-tRNA, consistent with their functioning as competitive inhibitors of peptide bond formation. Two hydrophobic crevices, one at the peptidyl transferase center and the other at the entrance to the peptide exit tunnel play roles in binding these antibiotics. Midway between these crevices, nucleotide A2103 of H. marismortui (2062 Escherichia coli) varies in its conformation and thereby contacts antibiotics bound at either crevice. The aromatic ring of anisomycin binds to the active-site hydrophobic crevice, as does the aromatic ring of puromycin, while the aromatic ring of chloramphenicol binds to the exit tunnel hydrophobic crevice. Sparsomycin contacts primarily a P-site bound substrate, but also extends into the active-site hydrophobic crevice. Virginiamycin M occupies portions of both the A and P-site, and induces a conformational change in the ribosome. Blasticidin S base-pairs with the P-loop and thereby mimics C74 and C75 of a P-site bound tRNA.

Introduction

Antibiotics are compounds of natural or synthetic origin that selectively kill or inhibit the growth of microorganisms, and many of them are sufficiently specific to be useful for the treatment of bacterial infections. A large fraction of these compounds, including many that are used in the clinic, are inhibitors of bacterial protein synthesis, and almost all such antibiotics interact with the ribosome. The peptidyl transferase center is the target of many of these antibiotics, which is surprising given the high-degree of conservation in that region of the ribosome, and given the need for antibiotics to target specific organisms. Since publication of atomic resolution structures of both the large and small ribosomal subunits,1., 2., 3. several papers have appeared describing the structures of complexes between ribosomal subunits and antibiotics.4., 5., 6., 7., 8. Here, we extend this series by reporting crystal structures of the complexes that form between the large ribosomal subunit of Haloarcula marismortui and five antibiotics: anisomycin, chloramphenicol, sparsomycin, virginiamycin M, and blasticidin S.

Anisomycin and sparsomycin are protein synthesis inhibitors that lack sufficient species-specificity for use in treatment of human infections, but have attracted interest in the context of cancer chemotherapy.9., 10. Chloramphenicol is highly specific for eubacteria and has a low-level of toxicity for humans. It is widely used, especially for eye infections.11 Blasticidin S is used to protect rice from fungal infection.12 Virginiamycin M (or streptogramin A), the last of the antibiotics considered in this series, is used to treat a broad range of infections.13 Virginiamycin M binds to ribosomes and inhibits translation by itself, but is more effective as a therapeutic13 when used in combination with virginiamycin S (streptogramin B) due to cooperative binding of these two compounds.14

Four of these antibiotics, anisomycin, chloramphenicol, sparsomycin, and blasticidin S, have structures suggestive of aminoacylated nucleosides. The prototypical aminoacylated nucleoside antibiotic is puromycin (Figure 1). Puromycin functions as an analogue of the 3′ end of a tyrosylated-tRNA15 and elongation is terminated when it becomes incorporated into a nascent peptide chain.16 Anisomycin and chloramphenicol have been described as structural analogues of puromycin17., 18., 19., 20., 21. because each of them is also comprised of a carbohydrate group attached to a phenyl group (Figure 1). Furthermore, blasticidin S consists of a cytosine bonded to a pyranose ring that has an amino acid-like appendage attached to it, and sparsomycin is a pseudouracil derivative. Therefore, it is not surprising that it has been suggested that all of these molecules might function as puromycin analogues.18., 19., 20., 22. The last member of the group, virginiamycin M, is completely unrelated to the others chemically (Figure 1). It is a streptogramin A, a class of compounds that includes a large heterocyclic ring.

The structures described here show that these antibiotics all bind near the peptidyl transferase center of the large ribosomal subunit at locations that should make their interaction with the ribosome competitive with the binding of tRNAs to either the A or P-site (Figure 2). In addition, all but blasticidin S interact with one of the two hydrophobic crevices in the peptidyl transferase region. Both crevices are formed by pairs of adjacent bases that stack imperfectly on each other so that a wedge-shaped hydrophobic gap exists between them. The ribosomal bases involved in the first crevice are A2486 (2451 Escherichia coli) and C2487 (2452), and that crevice normally interacts with the amino acid side-chains of aminoacyl-tRNAs bound in the A-site.23 The second crevice is formed by G2099 (A2058) and A2100 (2059), and is located at the entrance of the peptide exit tunnel. In addition to interacting with some of the antibiotics discussed here, this crevice also plays an important role in macrolide binding to the ribosome.7

Section snippets

Structure determination

The crystal structures of the complexes that the 50 S ribosomal subunit of H. marismortui forms with anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M were solved by X-ray crystallography at 3.0 Å resolution (see Materials and Methods). In each case, the location, orientation, and conformation of the bound antibiotic was unambiguous in the difference electron density maps (Figure 3). The structure of each antibiotic was fit into its corresponding electron density by

The active-site hydrophobic crevice

The tendency of chemical groups belonging to antibiotics to interact with the hydrophobic crevice in the peptidyl transferase active-site appears to be an important and general principle for small molecule-RNA affinity (Figure 4). Antibiotics that have aromatic groups tend to interact with it by inserting an edge of the aromatic group, but the details of the geometry vary significantly. For example, the aromatic rings of both puromycin23 and chloramphenicol8 approach the crevice in the same way

Conclusions

Two hydrophobic crevices near the peptidyl transferase center interact with many antibiotics. These crevices border opposite sides of an oblong pocket. One crevice is at the peptidyl transferase center and the other is at the entrance to the peptide exit tunnel. A2103 (2062) is midway between these crevices, and depending on its conformation can interact with ligands that are bound to either crevice. Hydrophobic and especially aromatic substituents of these antibiotics insert into these

Materials and Methods

Ribosomes were purified and crystallized as described previously.1 Anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M were purchased from Sigma. Virginiamycin S was a generous gift from Pfizer Pharmaceutical, and was soaked into crystals with virginiamycin M. Antibiotics were initially dissolved in dimethyl sulfoxide (DMSO) and then added to the standard stabilization buffer at concentrations from 1.0 mM to 10 mM and at final DMSO concentration of 1–4% (chloramphenicol

Acknowledgements

We thank Joe Ippolito for help with chloramphenicol; Betty Freeborn for her expertise in preparing both 50 S ribosomal subunits and crystals, and for useful discussions; Joyce Sutcliffe and Debra Tumbula-Hansen for thoroughly reading and commenting on the manuscript; Nenad Ban and Poul Nissen for work on the early stages of this project; Jimin Wang for useful discussions about crystallographic software; Martin Schmeing, Dan Klein, and Satwik Kamtekar for help with data collection; Andres

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